1. Field of the Invention
The present invention relates to a composition for preventing protein degradation, which contains small heat shock proteins (sHSPs), as well as a composition for use in two-dimensional (2-D) gel electrophoresis. Moreover, the present invention relates to an improved method of 2-D gel electrophoresis, which is characterized by using the sHSPs.
2. Background of the Related Art
As the base sequence of a human genome is revealed and genome information for numbers of microorganisms, lower animals and plants increases daily, proteomics becomes the focus of the next-generation research.
The proteomics that is a science field for studying proteomes systemically is distinguished from genomics. The proteomes signify complete information for the kind and amount of proteins, which are expressed from genomes under specific condition. Thus, the proteomics simultaneously analyzes and identifies various proteins in cells or tissues that are involved in biological phenomenon. Since this proteomic analysis provides results that cannot be found in genome projects or DNA researches, there are studies being conducted to develop diagnostic reagents or therapeutic agents for adult diseases, such as cancer, diabetes, dementia, and heart and circulation system diseases, and mental diseases, using this analysis, and also studies to apply it in fields, such as organ transplantation.
Core technology that has most widely been used in proteomics studies is a 2-D gel electrophoresis technique. The 2-D gel electrophoresis technique is the best method capable of separating and quantifying total proteins in cells or tissues.
The 2-D gel electrophoresis technique is a method where a mixture of proteins is first separated according to the isoelectric point (pI) of each protein, and each of the separated samples is further separated according to its molecular weight in a vertical direction such that the separated proteins are 2-Dly distributed on a plane. Namely, an isoelectric-focusing (IEF) method and a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) method are used respectively.
Currently, 2-D gels using IEF were developed, and commercialized systems appeared one after another, to greatly improve reproducibility that is a problem of the prior 2-D gel (U.S. Pat. No. 6,554,991; U.S. 2002/157954; U.S. 2002/133300; U.S. Pat. Nos. 6,416,644; 6,398,932; WO 02/25259; U.S. 2001/032786; U.S. 2001/023826; U.S. 2001/015320; U.S. Pat. Nos. 6,245,206; 6,136,173; 6,123,821; 5,993,627; WO 98/59092; and WO 02/90966). Furthermore, the steps of staining individual proteins in the 2-D gel and digesting the stained proteins with protease were preformed using an automated system and a computer so that samples could be processed in an easy and simple way.
However, the automation of the 2-D gel electrophoresis which is the first step is not yet realized. Moreover, since there is protein loss in all the process of the 2-D gel electrophoresis, it is impossible to completely analyze complex proteomes in cells or tissues. If cells are lysated in a first step, as protease is released from the cells protein degradation occurs to reduce the total number of proteins.
For this reason, a variety of the following methods for inhibiting protease attack in a protein separation process were designed: (1) the direction addition of strong denaturants to samples; (2) the preparation of samples at a low temperature or an alkaline condition (above pH 9); and (3) the use of protease inhibitor. Examples of the protease inhibitor include phenylmethyl-sulphonyl fluoride (PMSF), aminoethyl benzylsufonyl fluoride or Pefabloc™ SC (AEBSF), ethylenediaminetetraacetic acid (EDTA), benzamidine, tosyl lysine chloromethyl ketone (TLCK), and tosyl phenylalanine chloromethyl ketone (TPCK). However, in such methods, proteolysis cannot be completely inhibited, and the kinds and origins of samples are very various such that an optimal process for preparing the samples should be empirically determined.
Meanwhile, sHSPs that are heat shock proteins (HSPs) with a low molecular weight of 15–30 kDa are induced by stress such as heat shock or the overproduction of certain proteins, and act to prevent protein denaturation. One or more of the sHSPs are present in each of all organisms from eukaryotes to prokaryotes, and the sHSPs known till now are given in Table 1 below.
TABLE 1The sHSPs known.OriginsHSPsAgrobacterium tumefaciens str. C58IbpA(U. Washington)Arabidopsis thalianaSHSPsBradyrbizobium japonicumHspB, HspH, HspC,HspFBrucella suis 1330IbpABuchnera aphidicola plasmid pBPS1sHSPsBuchnera aphidicola str. APSIbpA(Acyrthosiphon pisum)Citrus tristeza virussHSPsEscherichia coli CFT073IbpA, IbpBEscherichia coli K12IbpA, IbpBEscherichia coli O157: H7 EDL933IbpA, IbpBEscherichia coli O157: H7IbpA, IbpBHelicobacter pylori 26695IbpBHumanHsp27, α, β-crystallinMethanococcus jannaschiiHSP16.5Methanopyrus kandleri AV19IbpAMurineHsp25Mycobacterium leprae strain TNsHSPsMycobacterium tuberculosisHsp16.3Pirellula sp.IbpBPisum sativum(pea)Hsp18.1Plasmodium falciparum 3D7sHSPsPseudomonas aeruginosa PA01IbpAPseudomonas putida KT2440IbpASaccharomyces cerevisiaeHsp26Salmonella enterica subsp. enterica serovar TyphiIbpA, IbpBSalmonella typhimurium LT2IbpA, IbpBShewanella oneidensis MR-1IbpAShigella flexneri 2a str. 2457TIbpA, IbpBShigella flexneri 2a str. 301IbpA, IbpBSinorhizobium meliloti 1021IbpASinorhizobium meliloti plasmid pSymAIbpAStreptococcus pyogenesIbpAStreptomyces coelicolor A3(2)sHSPsSulfolobus solfataricussHSPsSynechococcus vulcanusHsp16Thermoanaerobacter tengcongensis strain MB4TIbpAThermoplasma acidophilumIbpAYersinia pestis KIMsHSPs, IbpA, IbpBYersinia pestis strain CO92IbpA, IbpB
Such sHSPs have a conserved region in an evolutionary process and thus has been performing substantially similar functions. ATP independent sHSPs perform a function of preventing protein aggregation irreversibly by combining with denatured proteins under heat stress condition. Therefore, ATP independent sHSPs return the denatured proteins to the original form by correct refolding in cooperation with ATP dependent HSPs(heat shock proteins). For example, it has been reported that IbpA derived from E. coli and IbpB derived from E. coli prevent a citrate synthase from being inactivated by blocking aggregation due to heat or oxidant (Kitagawa et al., Eur. J. Biochem., 269:2907–17, 2002). It has been reported that HSP18.1 derived from pea has a function of blocking aggregation of proteins, such as malate dehydrogenase(MDH), glyceraldehydes-3-phosphate dehydrogenase, etc. under heat stress condition (Lee et al., EMBO J., 16:659–71, 1997). It has been reported that sHSPs derived from Bradyrbizobium japonicum has a function of blocking aggregation of citrate synthase due to heat (Studer and Narberhaus, J. Biol. Chem., 275:37212–8, 2000). It has been reported that a α-crystallin derived from human helps correct refolding of target proteins, which is denatured due to heat stress, by preventing aggregation in the process of dialysis (Horwitz, J., Proc. Natl. Acad. Sci. USA, 89:10449–53, 1992). Besides, a Pfu-sHSP purified from heat stable organism stabilizes Taq polymerase and enzyme at the high temperature because it protects cell proteins under heat stress condition in the process of PCR (WO 01/79250 A1). Moreover, sHSP 25 derived from Murine stabilizes unstable proteins or peptides in a diagnostic assay (Ehrnsperger et al., Anal. Biochem., 259:218–25, 1998). However, it was not yet known that these sHSPs prevent protein degradation.
Accordingly, the present inventors have conducted intensive studies to develop a method for preventing proteins from being degraded upon 2-D gel electrophoresis, and consequently, first found that the sHSPs had the effect of preventing protein degradation, and also if 2-D gel electrophoresis is performed using such sHSPs, gels with a significantly increased number of protein spots could be obtained, thereby achieving the present invention.